1. Field of the Invention
Acidovorax avenae, formerly classified as Pseudomonas avenae, is a seedborne pathogen of several hosts including oat, corn, millet, wheat, sugarcane, rice, and melon (watermelon and cantaloupe). This invention relates to novel PCR primers which can be used to detect plant pathogenic Acidovorax avenae subspecies and to distinguish among the pathogenic subspecies, particularly to distinguish A. avenae subsp. citrulli from other subspecies.
2. Description of the Relevant Art
The bacterium A. avenae subsp. avenae causes bacterial stripe of rice, leaf blight of oats, red stripe disease of sugarcane and millet, and brown stripe of Setaria italica. The organism was originally described as Pseudomonas avenae by Manns in 1909 (Ohio Agric. Exp. Stn. Res. Bull. 210: 91-167) as the causal agent of leaf blight on oats in Ohio. Another organism, P. alboprecipitans, described by Rosen in 1922 on foxtail (Ann. Mo. Bot. Gard. 9: 333-402) and on corn in 1926 (Phytopathology 16: 241-267) was later shown to be synonymous with P. avenae (Schaad et al. 1975. Int. J. Syst. Bacteriol. 25: 133-137). On rice, the organism has been previously referred to as P. setariae or P. panici (Goto. 1964. Bull. of Faculty of Agriculture, Shizuoka University 14: 3-10) and on sugarcane as P. rubrilineans (Martin et al. 1989.), the causal agent of red stripe diseases of sugarcane (C. Ricaud et al. (Eds), Elsevier, The Netherlands, pp. 80-95). Acidovorax avenae subsp. citrulli causes bacterial blight and fruit blotch of watermelon and melon. The organism was originally described by Schaad et al. (1978. Int. J. Syst. Bacteriol. 28:117-125) in plant introduction breeding lines of watermelon in Georgia as P. pseudoalcaligenes subsp. citrulli.
Phylogenetically, A. avenae is included in the new "acidovorans" DNA-rRNA homology group (Willems et al. 1992. Int. J. Syst. Bacteriol. 42: 107-119). This group contains many pathogens previously classified in the non-fluorescent pseudomonad group, including P. avenae Manns 1909 (Ohio Agric. Exp. Stn. Res. Bull. 210: 91-167), P. cattleyae Savulescu 1947 (Pavarino. 1911. Atti Accad. Lincei 20:233-237), P. pseudoalcaligenes subsp. citrulli (Schaad et al. 1978, supra), and P. rubrilineans (Lee et al. 1925. Red Stripe Disease. Pamphlet of Hawaiian Sugar Planters Association). Recently, all of these bacteria have been reclassified as subspecies of A. avenae, i.e., A. avenae subsp. avenae, A. avenae subsp. cattleyae, and A. avenae subsp. citrulli, on the basis of the results of DNA--DNA hybridization, DNA-rRNA hybridizations, polyacrylamide gel electrophoresis of whole-cell proteins, and a numerical analysis of carbon assimilation tests (Willems et al., supra).
Presently, the only reliable method to identify the A. avenae pathogens, is to isolate them on agar media and confirm their presumptive identification by time consuming and expensive pathogenicity tests. For example, in rice, A. avenae subsp. avenae can be recovered from both diseased, or asymptomatic, apparently healthy seeds. However, identification is difficult, because the organism is frequently overgrown by other bacterial pathogens of rice, such as Pantoea (Erwinia) herbicola, Burkholdera (Pseudomonas) glumae, B. fuscovaginae, or P. syringae pv. syringae. These pathogens are recognized as producing distinct disease symptoms, but field diagnosis is very difficult. In such cases, detection and identification is based on pathogen isolation, through the use of semiselective media, and pathogenicity tests which discriminate between the various pathogens. However, the presence of coexisting epiphytes such as P. putida and P. fluorescens in/on rice seed makes isolation difficult. Furthermore, rice seedlings are difficult to grow and symptoms on seedlings are often non-discriminating. Several methods of identification of A. avenae subsp. avenae have been proposed including growth and isolation on semiselective agar media and serology (Shakya. 1987. Korean J. Plant Path. 3:300; Zeigler et al. 1989. Intl. Rice Res. Newsletter 14: 27-28). None have achieved much success. There is currently no reliable, routine method available for detecting A. avenae in rice seeds. More sensitive and specific methods are needed to confirm the identification, especially in seed health evaluations.
Similar problems exist for melons. Although originally described as a seedling disease, watermelon fruit blotch has emerged as a serious disease of mature fruit. Complete losses of production fields often occur due to fruit rot. Like most seedborne bacteria, control is based primarily on seed health testing. Because of the seriousness of watermelon fruit blotch, nearly all watermelon seed lots must be assayed for A. avenae subspec. citrulli. This involves soaking seeds and attempting to isolate the organism on agar media or inoculating plants with the seed soakate. Neither method is very efficient and both are relatively expensive. Little or no resistance to the pathogen exists.
Bacterial stripe causes great losses in rice seedling beds throughout Asia. Thus far, the disease has not been observed in the U.S. However, the potential risk of the dissemination of the bacterial stripe pathogen in international exchange of germplasm of rice, and also of corn and watermelon, is a serious concern. There is presently no way to separate infected seeds and contaminated seeds. Therefore, there is a need to develop reagents and methods for detecting A. avenae subspecies specifically, rapidly, and directly from biological samples. Such methods and reagents are valuable tools for monitoring natural disease spread, tracking the seedborne bacteria in field studies, and detecting the presence of the bacterium in seed lots entering A. avenae -free areas.
Polymerase chain reaction (PCR) has been shown to be highly sensitive and the method is commonly used to detect and identify bacteria. A PCR method has been described for detecting the pathogen A. avenae subsp. citrulli, in seeds; however, the primers are not unique and the method has not gained industry acceptance (Minsavage et al. 1995. Ann. Mtg. Amer. Phytopath. Abstract 379). Thus, there exists a need for specific primers and methods capable of specifically identifying and differentiating pathogenic A. avenae subspecies.